Sexual transmission of HIV is by far the predominant means for the spread of the virus. In the absence of an effective vaccine, anti-HIV topical microbicides offer a relatively inexpensive means to minimize HIV transmission. The ideal anti-HIV-1 microbicide should fulfill several requirements. It must act directly on a wide variety of HIV strains and subtypes to inactivate the virus and prevent subsequent infection. It should act at replication steps prior to integration of proviral DNA into the infected host cell genome. It should be non-toxic and absorbable by uninfected cells and localize within these cells, to provide a barrier to infection by residual active virus. Finally, while the microbicide should be absorbable, it must not readily transit the cell, to prevent systemic absorption of the drug by healthy individuals. UC781, a tight-binding non-nucleoside reverse transcriptase inhibitor (NNRTI) has been shown to possess potent anti-HIV-1 microbicidal activity. However, it is unlikely that any single compound is sufficient to provide high-level protection against HIV transmission; combination microbicides are preferable. This project will evaluate the in vitro microbicide activity of UC781 in combination with a variety of other antiviral agents, including octyl glycerol (a surfactant that disrupts the HIV membrane envelope), carrageenan (which inhibits HIV-cell interaction) and bioflavonoids (which irreversibly inhibit gp120, and also inhibit RT). The Specific Aims of the project include: (1) evaluation of the in vitro microbicidal properties of UC781, other active antiviral components, and "inactive" excipients, alone and in combination. This will involve testing against a variety of HIV isolates (laboratory-adapted strains, primary isolates including different subtypes, etc.) and against different cell types implicated in the mucosal transmission of HIV (lymphocytic cells, macrophages, epithelial cells, and dendritic cells; (2) to determine whether microbicides with UC781, alone and in combination with other active antiviral components, will select in vitro for transmission of NNRTI-resistant virus; and (3) to develop and validate a sensitive analytical method for the quantitation of UC781 in blood, plasma, and tissue, and to use this method to quantitate UC781 levels in samples from human clinical trials (Project 4). Our studies will predict appropriate combinations and concentrations of active excipients for use in initial microbicide formulations, which will be tested and modified based on data from all Program Project components, in an iterative manner, to provide an optimal microbicide formulation to prevent the sexual transmission of HIV.